Kaličanin Nevena, Balaž Ana Marija, Prodanović Olivera, Prodanović Radivoje
University of Belgrade-Institute of Chemistry Technology and Metallurgy National Institute of the Republic of Serbia, Njegoševa 12, 11000, Belgrade, Serbia.
Department of Otorhinolaryngology Head and Neck Surgery, Grosshadern Medical Center, Ludwig-Maximilians-University, Marchioninistr. 15, 81377, Munich, Germany.
Chembiochem. 2023 Oct 17;24(20):e202300414. doi: 10.1002/cbic.202300414. Epub 2023 Aug 22.
The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0-11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide.
本研究的目的是证明来自雷特巴盐脱硫嗜盐菌的假定的opine脱氢酶的功能，并根据功能和动力学参数对该酶进行表征。通过基于序列的宏基因组文库技术搜索从宏基因组文库中鉴定出一种假定的opine脱氢酶，随后在大肠杆菌中成功实现了该酶的异源表达。为了研究其在仲胺合成中的应用潜力，首先测定了该酶对不同氨基供体和氨基受体的底物特异性。观察到该酶对小氨基酸具有最高亲和力，优先选择L-丙氨酸，而对于α-酮酸，丙酮酸被证明是优先的氨基受体。在无盐条件下，pH 6.5时观察到最高活性。该酶在广泛的实验条件下表现出显著的稳定性，如宽泛的pH稳定性（在特定pH缓冲液中孵育30分钟后，pH范围为6.0 - 11.0）、在高达3.0 M的NaCl存在下24小时的稳定性、在45°C孵育1小时后保留80%的初始活性以及在30%二甲基亚砜中孵育24小时后保留65%的初始活性。
